Biology Reference
In-Depth Information
of laminin fragments. In recent years, several new baculovirus expression systems
have been developed and proven to be equal to those described herein.
4.
We use the BaculoGold transfection kit for simplicity and ease of isolation of
recombinant clones. We have substituted unsupplemented Grace's medium,
pH 6.1 (Gibco-BRL; 11590-056) for transfection Buffer A and sterile-filtered
25 m
M
HEPES, 140 m
M
NaCl, 125 m
M
CaCl
2
, pH 7.1 for transfection Buffer B
and purchased BaculoGold DNA rather than the complete transfection kit. Tradi-
tional methods of calcium phosphate precipitation with wild-type AcMNPV virus
have also proven useful, but require longer periods of time to isolate recombinant
virus that exclude AcMNPV that has not undergone homologous recombination.
Transfections using cationic lipids such as Cellfectin (Gibco-BRL; 10362-010)
have also been successful for isolation of recombinant baculovirus from infected
Sf9 cells.
5.
Grace's insect cell culture medium (Gibco-BRL; 11605-094) is an alternative
medium for cell culture.
6.
Serum components have been known to vary with lot number. As with all cells
grown in culture, it is advised to screen several lots of serum for optimal cell growth,
transfection efficiency, and recombinant protein production and stability.
7.
The major problem encountered with growth of Sf9 cells in suspension is aera-
tion. Insufficient aeration can give rise to decreased cell viability and subsequent
release of cytosolic proteinases upon cell lysis. Spinner flasks should be main-
tained at or near 100 rpm and never be filled above two-thirds of flask volume.
Circulation greater than 100 rpm may result in additional cell death by “beating
the cells to death”. Should cell viability fall below 95% given these culture
conditions, decrease the flask volume to 1/2 and/or add surfactants such as
Pluronic F-68 (BASF Corp.; 588280).
8.
We prefer to use this method for the concentration of proteins in dilute solution
to preserve native functions; it is the gentlest means of protein concentration.
Traditional methods of such as lyophilization, salting out, or acid/acetone
precipitation may irreversibly denature some proteins, especially a concern if
conformational stability is required for function. Caution should be exercised so
as to not over-concentrate proteins in solution because of the risk of precipita-
tion, and should never be allowed to dry in dialysis tubing.
9.
Sf9 cells can be weaned of serum-requirements for growth by twofold reduction
of serum content with passaging the cells. A minor lag phase in cell division is
usually evident after the first three serial dilutions, but later is overcome through
adaptation to reduced serum content in the growth medium. We have found that
Sf9 cells grown in suspension are more easily weaned of serum-dependence than
those grown as monolayers.
10.
DNA precipitation is usually evidenced by turbidity.
11.
It had been suggested that recombinant baculovirus is light-sensitive; hence, we
typically store viral stocks in the dark or wrapped in aluminum foil to reduce
exposure to light. Long-term storage of recombinant baculovirus at 4
C is not
recommended because viral titer decreases with prolonged storage. Aliquots of
°
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